Project Summary Each herpesvirus encodes a viral homolog of mammalian uracil DNA glycosylase (vUNG). Mammalian UNG is an enzyme that removes misincorporated and mutagenic uracils, leaving an abasic site typically repaired by the host base excision repair pathways. Although there is conserved sequence and in vitro activity between vUNG and host UNG, little is known regarding vUNG function in the virus lifecycle or effect on host genome DNA repair. Studies indicate that herpesvirus vUNG may interact with components of the viral DNA replication machinery; however, its role in late stage replication events in primary cells is uncertain. We utilize murine gammaherpesvirus 68 (MHV68) as a mouse model for human gammaherpesviruses, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Our studies indicate that herpesvirus vUNG plays a critical role in pathogenesis, as vUNG deficient virus has major replication and infectivity defects. Gammaherpesvirus infects germinal center (GC) B cells and establishes latency in memory B cells, a process that can lead to immortalization and transformation of B cells. In GC B cells, host UNG activity process AID induced uracils to trigger antibody diversification, a process that also triggers chromosome translocations that are associated with lymphomas. Our studies indicate vUNG can process uracil in genomic DNA but with repair outcome different than host UNG. vUNG suppresses antibody mutation and class switch recombination in a manner that is both catalytic and non-catalytic dependent suggesting a scaffold function for vUNG. Thus, vUNG has the potential to contribute to altered viral pathogenesis, immunity and malignancy. We propose studies to understand how vUNG promotes viral pathogenesis and subverts host antibody diversification. In Aim 1, the structural and biochemical basis for error-free repair of DNA lesions by vUNG will be investigated. Differences between vUNG and host UNG will be characterized to determine the domains and interacting partners that result in differential error-free versus error-prone repair outcome. In Aim 2, the mechanisms by which vUNG promotes gammaherpesvirus pathogenesis will be investigated. The contribution of vUNG enzymatic function and scaffold functions will be characterized. In Aim 3, the impact of gammaherpesvirus infection and vUNG on B cell development will be investigated. B cell activation and immunoglobulin repertoire will be examined during MHV68 infection. Antibody repertoire and self-reactivity will be analyzed during acute infection and latency reactivation. Our aims straddle molecular immunology and molecular virology in the fields of gammaherpesvirus pathogenesis and B cell biology. The proposed studies should elucidate the mechanism by which vUNG supports viral replication, infection, and viral subversion of immunoglobulin diversification.